The differential expression of cell surface immobilization (i-) antigens of ciliate protists is an old probelm in developmental genetics. Similar phenomena exist in Trypanosomes (causative agents of Chagas Disease) and in cancer and tissue cells of mammals. The cell surface not only bears the markers of differential gene expression, but itself mediates signals that change the state of gene expression. The i-antigens of Tetrahymena thermophila permit both biochemical and genetic studies of cell surface proteins and as such provide a convenient model for eukaryote gene regulation. T. thermophila has the genetic information to express at least six "mutually exclusive" i-antigens, but normally only one i-antigen is present on the cell surface. Disturbance of the cell surface causes the expression of an alternate i-antigen. The goal of this proposal is the exhaustive mutational analysis of the i-antigen system. The hypothesis is that i-antigens are regulated both by regulator genes, some of which have already been identified, as well as by the i-antigen molecules themselves, for which tentative evidence exists. New genetic technology, namely cytogamy and newly created nullisomics and deletion homozygotes (the first ever in ciliates) to undertake a chromosome by chromosome (N=5) mutational study of both the regulator and structural genes. Further work on the purification and characterization of i-antigen molecules and the development of an in vitro translation system are also proposed. This project is part of an ongoing effort to understand completely one system of differential gene expression in an eukaryote. Subsequent studies will undertake cloning and sequencing genes identified by mutational analysis. The mutational analysis is necessary first to identify as many as possible of the regulator genes and elements controlling i-antigen expression. A unique feature of i-antigen regulation is that it takes place in a compound macronucleus. This project, therefore, offers important comparisons and contrasts to gene regulation in diploid cells.